Bioresorable polymer matrices and methods of making and using the same

ABSTRACT

Bioactive agents are delivered to a body site in need of the same by providing a first aliquot portion of a reaction mixture which includes an aldehydic polymer solution, and a second aliquot portion of a reaction which includes a cross-linking hydrazide and a bioactive agent. The first and second aliquot portions may be mixed (e.g., by expelling such portions from respective syringe barrels) to form the reaction mixture thereof. The thus formed reaction mixture may then be installed at the body site whereby the reaction mixture is allowed to solidify in situ within about 1 to 10 minutes into a reaction product comprised of a hydrazide cross-linked oxidized aldehydic polymer matrix with the bioactive agent entrapped therein.

CROSS-REFERENCE

This application is a continuation of commonly owned U.S. Ser. No.13/867,929, filed Apr. 22, 2013 (U.S. Pat. No. 8,664,280) which is acontinuation of U.S. Ser. No. 13/228,267, filed Sep. 8, 2011 (U.S. Pat.No. 8,435,565) which is a continuation of U.S. Ser. No. 11/983,454,filed on Nov. 9, 2007 (U.S. Pat. No. 8,039,021), which is a continuationof PCT/US2006/02380, filed May 26, 2006, which is based on and claimsdomestic priority benefits under 35 USC §119 (e) from U.S. ProvisionalApplication Ser. No. 60/684,974, filed May 27, 2005, the entire contentof each application being hereby incorporated by reference.

FIELD OF INVENTION

The present invention relates generally to bioresorbable polymermatrices and to their production and use as delivery systems forbioactive agents. Sustained and/or controlled release of pharmaceuticalsand other bioactive agents, including cells, are the primary uses of thematrices of the present invention.

BACKGROUND AND SUMMARY OF THE INVENTION

Polymer matrices designed for controlled release of bioactive compoundscan be non-resorbable or resorbable. In general, resorbable meansbiologically degradable in the body by erosion from the surface ofbreakdown from within. The mechanism can involve either a chemicalreaction, such as hydrolysis, or dissolution. Non-resorbable polymers,such as polymethylmethacrylate, have been used for antibiotic delivery.These materials suffer from the disadvantage that they must beretrieved, which involves a second intervention and entails the risk ofinfection (H W Bucholz, et al., (1970) Chiburg, 43, 446).¹ ¹ Thispublication as well as other publications cited hereinbelow areexpressly incorporated in their entirety into this application.

Preparation of oxidized dextran is described in U.S. Pat. No. 5,783,214to Royer. Oxidized hyaluronic acid has been used in reactions with aminopolysaccharides to form complexes as described in U.S. Pat. No.6,305,585 to Spiro et al; however, the disclosed method is lessconvenient and more expensive compared to the system described herein.Also, the cross-linking conditions require uncharged amino groups, whicheliminates many active ingredients, as they would react with thepolymer. The gel described in U.S. Pat. No. 6,305,585 is prepared inbulk by reacting two polymer solutions at alkaline pH. Lee et al,Macromolecules, 33, 97-101 (2001), have described a structuralbiomaterial made of poly(guluronate), which involves an oxidized polymerisolated from alginic acid. The starting material is not commerciallyavailable, is expensive to produce, and has not been FDA-approved formedicinal Use. The production of this biomaterial occurs at high pH thatwould involve reaction of amino groups in proteins or of other medicinalcompounds.

Another polysaccharide that finds application in medicine iscarboxymethylcellulose used in drug formulation and also the productionof anti-adhesion compositions for post-surgical treatment followingseveral types of procedures (Schwartz H E and Blackmore J M, U.S. Pat.Nos. 6,017,301, 6,133,325, and 6,034,140; Miller M E, Cortese, S M,Schwartz, H E and Oppelt, W G, U.S. Pat. No. 6,566,345; Llu, L S andBerg, R, U.S. Pat. No. 6,923,961; Schwartz, H W, Blackmore, J M,Cortese, S M, and Oppelt, W G, U.S. Pat. No. 6,869,938). Theseformulations involve the non-covalent aggregation of carboxylatedpolysaccharides with polyethyleneglycols.

Hyaluronic acid has been used as a biomaterial for a number ofapplications, Gertzman and Sunwoo have employed the sodium salt of highmolecular weight hyaluronic acid to deliver demineralized bone matrix(U.S. Pat. Nos. 7,019,192; 6,911,212; 6,458,375 and 6,328,018). Thematerial used is a solution and does not involve chemical cross-linkingas described herein. The covalent cross-linking provides for gelstability and retards migration of the bioactive agent. Also, the degreeof cross-linking constitutes an element of control for the residencetime/resorption rate.

In contrast to some of the polymers cited in the above references,dextran employed in the practice of the present invention is availableas a USP product and has been used parenterally for many years. Acrylatederivatives of dextran have been produced and can form cross-linkedhydrogels (De Groot et al, (2002) Int. J. Cancer, 98, 134-140; Stenekes,et al. (2000) Biomacromolecules, 1, 696-703; Chung, et al. (2005) Int.J. Pharm., 288, 51-61). Polylactide-dextran grafts can be produced andemployed to make a delivery matrix using an emulsion based microspheresystem (Quchi, et al. (2004) 4, 455-463)

Commonly owned international Patent Publication No. WO 2004/112712discloses novel drug polymer complexes, which are formed by mixing apolyanion such as dextran sulfate sodium and a cationic bioactive agent.The resulting resorbable gels (R Gels) or precipitate constitutes a drugdelivery matrix. As disclosed, however, there are only ionic bondsinvolved in the formation of such R Gels. Furthermore, the formation ofsuch R Gels requires that the active pharmaceutical ingredient must bepositively charged. Many active ingredients, however, are neutral ornegatively charged at physiological pH. The carbohydrate matrix of RGels is very attractive in that it is polar and not susceptible toproteolytic degradation.

It would therefore be highly desirable if the resorbable polymer systemswere able to deliver neutral or negatively charged active ingredients atphysiological pH. It is towards providing such resorbable polymersystems that the present invention is directed.

Broadly, the present invention disclosed herein entails the usage ofchemically modified polysaccharides in the preparation of deliverymatrices for drug molecules, which are not usable with conventional RGels, included are negatively charged and neutral active ingredients. Inespecially preferred embodiments, the matrix involves cross-linking ofaldehydic polymers with dihydrazides in the presence of an activeingredient. The cross-linking reagent, the dihydrazide, reacts to form ahydrazone. A film, microbeads, or rubbery solid is the product of thereaction.

According to one aspect of this invention, bioerodible compositions fordelivery of a bioactive agent are provided which are the reactionproducts of a reaction mixture comprised of an oxidized dextransolution, and a mixture of solids comprised of a dihydrazide, abioactive agent, and optionally a pH adjusting agent in an amountsufficient to achieve a pH of the reaction mixture of 6 or less.Preferably the pH of the reaction mixture will be 6 or less whether ornot a pH adjusting agent is employed. If employed, however, preferred pHadjusting agents are solid acids, such as sodium phosphate, citric acid,succinic acid and/or fumaric add.

The compositions of the invention may further comprise a release agentfor controlling release of the bioactive agent from the composition. Onepreferred form of the release agent is a complexing polymer which bindsto the bioactive agent. Alternatively or additionally, the release agentmay comprise a non-reactive matrix polymer to provide structuralstability and diffusional resistance to the bioactive agent, In somepreferred embodiments of the invention, the release agent will comprisesan aliphatic or aromatic acid which forms salts with aminoglycosideantibiotics, vancomycin, tetracyclines or clindamycin.

Virtually any bioactive agent may be employed satisfactorily in thepresent invention. In this regard, preferred bioactive agents compriseosteoinductive agents, antibiotics, anesthetics, growth factors, cells,anti-tumor agents, anti-inflammatory agents, antiparasitics, antigens,adjuvants and cytokines. One particularly preferred bioactive agent isan osteoinductive agent, such as demineralized bone matrix (DBM).

The compositions of the invention may be provided in the form of a gel,to microsphere, film, foam, or fiber.

According to another aspect of this Invention, kits for forming abioerodible composition adapted for delivery of a bioactive agent areprovided. The kits of the invention will comprise a first aliquotportion of a reaction mixture comprised of an oxidized dextran solution,and a second aliquot portion of a reaction mixture comprised of amixture of solids which include a dihydrazide, a bioactive agent, andoptionally a pH adjusting agent in an amount sufficient to achieve a pHof the reaction mixture of 6 or less. When mixed the first and secondaliquot portions of the reaction mixture react to form a solidifiedbioerodible composition for delivery of the bioactive agent containedtherein. The release agent, if employed, may therefore be incorporatedinto one of the first and second aliquot portions of the reactionmixture.

The kits of the invention may advantageously be provided with a doublesyringe having first and second syringe barrels which respectivelycontain the first and second aliquot portions of the reaction mixture.

According to yet another aspect of the invention, methods of making asolidified bioerodible composition for delivery of a bioactive agent areprovided. The preferred methods comprise forming a reaction mixturecomprised of an oxidized dextran solution, and a mixture of solidscomprised of a dihydrazide, a bioactive agent, and optionally a pHadjusting agent in an amount sufficient to achieve a pH of the reactionmixture of 6 or less, and allowing the reaction mixture to react to forma solidified bioerodible drug delivery composition.

Several systems may be employed to prepare the resorbable polymer matrixaccording to the invention for administration. For example, it ispossible to connect two syringes physically one to another and to effectmixing of the reactable components by reciprocating action no as totransfer the mixture repetitively from one syringe barrel, to another.The pH is held at 6 or below to prevent reaction of amino groups on theactive ingredient. The bioactive agent, dihydrazide, and solid acid (alldry powders) are contained in the first syringe and the oxidized dextransolution in the in second syringe. There are many advantages to thisformat including stability of the bioactive agent, maintenance ofsterility, and convenience.

In yet another aspect, the present invention is embodied. In implantablemedical device which comprises a coating of the solidified bioerodiblecomposition. The medical devices may advantageously be in the form of acatheter, a stent or an orthopedic device. Particularly preferred coatedorthopedic devices in accordance with the present invention includepins, screws, rods, nails, wires, augments, cups, and joint implants.

According to yet another aspect of the present invention, methods ofdelivering a bioactive agent to a body site in need of the same areprovided. The methods comprise providing a first aliquot portion of areaction mixture comprising an oxidized dextran solution, and a secondaliquot portion of a reaction mixture comprising a mixture of solidscomprised of a dihydrazide, a bioactive agent, and optionally a pHadjusting agent in an amount sufficient to achieve a pH of the reactionmixture of 6 or less. The first and second aliquot portions may be mixedtogether to form the reaction mixture, so that subsequently, thereaction mixture may be installed at the body site and allowed to form asolidified bioerodible drug delivery composition to be formed thereby insitu. A double syringe having first and second syringe barrels whichrespectively contain the first and second aliquot portions of thereaction mixture may be provided. The step of expelling the first andsecond aliquot portions of the reaction mixtures may therefore bepracticed by repeatedly transferring the first and second aliquotportions of the reaction mixtures between the first and second syringebarrels so as to mix the same.

These and other aspects and advantages will become more apparent aftercareful consideration is given to the following detailed description ofthe preferred exemplary embodiments thereof.

DETAILED DESCRIPTION OF THE INVENTION

The components necessarily required to producing the polymer complexesof the present invention include an bioactive agent, an aldehydicpolymer, and a multifunctional hydrazide. Mixing the above componentsresults in a solid matrix as a consequence of the reaction ofdihydrazide with the aldehyde groups on the polymer chains with threedimensional cross-linking. The reaction is run at acid pH (e.g., betweenabout 4.0 to about 6.0) In order to speed the gel formation and toprevent reaction of amino groups of the active ingredient. The activeingredient is thus trapped within the resulting polymer matrix. Shortlyafter mixing the components the solution/suspension isinfected/installed in an animal or human patient. The matrix solidifiesin situ within several minutes (e.g., usually within about 5 minutes).The hydrated polymer matrix degrades in viva and is therefore resorbableby the body. Unlike polyesters no acid is produced as a result ofresorption.

Non-reactive polymers may be included to alter the diffusionalresistance or to complex the bioactive agent.

In general, the ideal drug delivery matrix displays the followingcharacteristics:

-   -   1. Non-toxic    -   2. Resorbable    -   3. Versatile as to molecular size and chemical nature of the        bioactive agent    -   4. No requirement far exotic components that are expensive or        difficult to obtain    -   5. Controllable release rate    -   6. Producible according to GMP at reasonable cost    -   7. Sterilizable

Disclosed herein are polysaccharide drug delivery matrices and themethodology for producing and using the same. More specifically, thepolysaccharide drug delivery matrices of the present invention may bederived from periodate-oxidized dextran and dihydrazides. Thecross-linking reaction is carried out at acidic pH, which allowsentrapment of bioactive substances with free amino groups. Protonationof the amino groups in the pH range of 4-6 renders such groupsrelatively unreactive as compared to the dihydrazides. Hence thebioactive agent remains in tact and does not consume aldehydiccross-linking sites.

It is important to maintain the pH of the reaction mixture to betweenabout 4.0 to about 6.0. In some cases, it may be necessary to add anagent, such as sodium phosphate or an organic acid (e.g., citric orfumaric acid), to adjust the pH to within the desired range. In thecases where the active ingredients are present as salts, the addition ofsodium phosphate or an organic acid to adjust the pH is not alwaysnecessary, in general; therefore, when the active ingredient contributesacidity, there may not be a need to add an acidic agent so as to adjustthe pH.

Vicinol diols react with sodium metaperiodate to yield aldehydes.Polyglucans, mannans, levans, and the like, react smoothly in aqueoussolution at room temperature. When a polymer such as dextran is reactedwith periodate the result is iodate and a polymer chain containingintermittent dialdehyde groupings. These dialdehydes serve as sites forreaction with adipic dihydrazide, which results in the formation of acovalently cross-linked hydrophilic gel.

The reaction to form the polymer complexes according to the presentinvention may be represented schematically as:

in which X represents the dihydrazone cross-link and D represents theactive ingredient. It should be stressed that only a fraction of thevicinol diols in the polysaccharide are reacted. On a residue molarbasis, the degree of oxidation is in the 5-20% range. The reactionscheme above is not intended to represent stoichiometry of the reaction.

Release rate and control of the release profile are importantconsiderations of drug delivery systems.

Fick's Law of diffusion states that:

Rate=DA(∂c/∂x)

where D is the diffusion coefficient; A is the surface area and (∂c/∂x)is the concentration gradient at the boundary of the matrix.

To paraphrase the Stokes-Einstein equation, D can be expressed as:

D=kS/M _(w) V

in which k is a constant, S is the solubility of the active ingredient,M_(w) is the molecular weight and V is the viscosity of the medium.Complexation can reduce the effective solubility and the addition ofnon-reactive polymers can increase the “viscosity” or diffusionalresistance. The oxidation level may be varied from between about 5% toabout 20%. The polymer concentration can be within the range of betweenabout 50 to about 250 mg/ml depending on the system employed forpreparing the formulation. Active ingredients can be converted tohydrophobic salts. For instance, amines can be converted with C6 orgreater carboxylic acids. These salts can be employed to extend therelease profile either alone or in combination with highly solubleinorganic salts.

The polymers to be oxidized must be biocompatible and resorbable. Anypolymer that meets these criteria may be used. Preferred are dextran(produced by microbial fermentation) and derivatives thereof; dextransfrom other sources may be employed as long as they are biocompatible andhave properties similar to USP dextran. The preferred molecular weightof the polymer is 40,000 or greater. In general, the reaction timerequired for gellation is longer when low molecular weight oxidizeddextran is employed.

The cross-linking hydrazides that are useful include the following:

Carbon No. Name

-   -   C4 Succinic add dihydrazide    -   C5 Glutaric acid dihydrazide    -   C6 Adipic acid dihydrazide    -   C7 Pimelic acid dihydrazide    -   C8 Suberic acid dihydrazide    -   C9 Azelaic acid dihydrazide    -   C10 Sebacic acid dihydrazide    -   C11 Undecanedioic acid dihydrazide    -   C12 Dodecanedioic acid dihydrazide    -   C13 Brassylic acid dihydrazide    -   C14 Tetradecanedioic acid dihydrazide    -   C15 Pentadecanedioic acid dihydrazide    -   C16 Thapsic acid dihydrazide    -   C18 Octadecanedioic acid dihydrazide

Organic solvent-water mixtures can be used to accelerate dissolution andreaction rates when higher molecular weight hydrazides are employed.

The matrices described herein are usefully employed for deliveringbioactive agents including, for example, drugs, hormones, cytokines,growth factors, cells, and the like. Direct injection for local orsystemic drug delivery is one mode of use. Others include coatings forimplants, wound sealant, topical wound dressing, local installationperioperatively for infection control or pain control (localanesthetic).

One embodiment of the present invention includes demineralized bonematrix (DBM) which is made by treating donor bone with acid to removethe inorganic components (Urist, M R, et al., Proc. Natl. Acad. Sci.(1984) 81, 372-375; Peterson, et al., JBJS (2004) 86, 2243-2250).Following sterilization, DBM can be mixed with a carrier and placed inan orthopedic defect. Osteoinduction results in new bone formation.

DBM can be delivered to an orthopedic defect using the matrix describedherein in at least two ways. First, the DBM dry solid can be blendedwith the dihydrazide and subsequently treated with oxidized dextransolution. The resulting liquid suspension is taken up in a syringe andinstalled by the surgeon. With most forms of DBM, the dual syringesystem can be employed.

Secondly, the mixture containing polymer matrix and DBM is allowed tosolidify. When dry, this material becomes quite hard and will bearweight. The dry composite is then fashioned to form the desired shape.Cancellous bone chips, hydroxyapatite, or other inorganic materials maybe included in the formulation prior to installation.

Those skilled in the art will of course recognize that it is possible tocombine other bioactive agents with the osteoinductive DBM. ADBM/antibiotic matrix may therefore be useful for treating bone andjoint infections.

Furthermore, a DBM/bupivacaine matrix may be useful for treating pain atbone graft procurement sites, such as the iliac crest, which is a commonsource for allograft bone for use in spine arthrodesis.

For a number of orthopedic applications, bone marrow aspirate is anotherlogical additive to use with osteoinductive DBM with the matrixdescribed herein.

Mesenchymal stem cells and other progenitor cells may be delivered withregulatory biochemicals. Chondrocytes may be delivered for repair ofarticular cartilage.

One additional beneficial use of the matrices according to the presentinvention is the prevention of post-surgical adhesions. Specifically,following surgery (such as abdominal, gynecological, or pelvic surgery)the installation of a matrix in accordance with the present inventionmay contribute to the significant reduction of adhesions. Variousadjuvants can be employed including fibrinolytic agents, anticoagulants,anti-inflammatory agents, and antibiotics. In this application anadditional polymer such as polyethyleneglycol (8000) is advantageouslyincluded in the oxidized dextran solution.

A duel syringe set-up may be used in preparation of the resorbablepolymer matrices according to the present invention. For example asolution of an aldehydic polymer, such as oxidized dextran, may becontained within the barrel of one syringe, while a mixture of soliddrug and solid dihydazide may be contained within the barrel of theother syringe. Monosodium phosphate may be included to control pH, whichis advantageously maintained in the range of between about 4.0 to about6.0. The syringes are connected to one another so that the contents maybe mixed by alternately transferring the mixture from one syringe barrelto another for about 30 cycles. Solidification occurs within about 1 toabout 10 minutes depending on the relative concentrations of components.The oxidized dextran solution is stable for at least one year and thesolid components are stable for at least that time, in some casesindefinitely. Maintenance of sterility, broad applicability, stabilityof the bioactive agent and ease of use are the attributes of such adouble syringe system.

Various physical forms of the invention may be produced. In this regard,it is especially preferred that the polymer matrices of the invention bein the form of an injectable liquid, which solidifies inside the body.The injectable liquid made with the double syringe system can also beapplied to surgical wounds/incisions or the wounds resulting frominjuries. The formulation solidifies and serves as a wound sealant.Antibiotics and growth factors may be used to prevent infection andpromote healing.

Another approach is to allow the dosage form to set up (solidify) exvivo prior to being administered. After drying and milling, theresulting powder can be used topically. Alternatively or additionally,the powder can be suspended and used parenterally.

A solid dosage form of the invention solidified ex vivo may be used as atopical anti-infective powder for wound treatment. Entrapment ofclindamycin and amikacin within a resorbable polymer matrix produces abroad spectrum, long lasting antibiotic powder. Other types ofantibiotics such as fluoroquinolones, glycopeptides, macrolides, betalactams and others can also be employed either singly or in combinationas may be desired.

Fibers of the resorbable polymer matrices according to the presentinvention can be prepared by injection spinning of the reaction mixtureinto isopropanol.

These fibers may be tailored by changing the relative amount ofcross-linking reagent and/or the use non-reactive polymers. The fiberscontaining antibiotic can be woven into a bandage or used as is to treatan infection such as a septic diabetic foot ulcer.

Microbeads are readily prepared using oxidized dextran and dihydrazides.After mixing using the double syringe the formulation is injected intorapidly stirred mineral oil containing a surfactant. The resultingmicrobeads are washed with organic solvent, dried, and packaged. Thesemicrobeads are suspendable in water and can be injected through a23-gauge needle.

The utility of the invention is further illustrated by the non-limitingentries shown in Table 1.

TABLE 1 Illustrative applications of the invention. Indication DosageForm Bioactive Agent Orthopedic Defects Injectable gel or DBM or otherImplantable solid osteoinductive agent Localized Infections Injectablegel, film, Tobramycin microspheres, fibers Clindamycin Pain ControlInjectable gel or Bupivacaine microspheres Regional Nerve BlocksInjectable gel or Bupivacaine microspheres Cancer Injectable gel orCarboplatin microspheres Paciltaxel Vaccine Injectable gel or Antigen,adjuvant microspheres

The present invention will be further understood after carefulconsideration is given to the following non-limiting examples thereof.

EXAMPLES Example 1 Preparation of Oxidized Dextran

Dextran (4 g; Mw 500,000) was dissolved in 20 ml distilled water. Tothis solution was added 535 mg of finely ground NaIO₄ with rapidstirring. The mixture was stirred in the dark for 1 hour and thendialyzed against three changes of distilled water (1 L each). Theoxidized dextran solution (about 70 mg/ml) was stored at roomtemperature in the dark. The solution was concentrated with a rotaryevaporator at 40° C. to about 200 mg/ml.

Example 2 Ciprofloxacin/Clindamycin Matrix

Oxidized dextran solution, prepared as described in Example 1 (1 ml, 93mg/ml), was loaded into a 3 ml syringe. A finely ground powdercontaining 21 mg adipic dihydrazide, 20 mg citric acid, and 150 mgciprofloxacin was loaded into a second 3-ml syringe. The syringe, whichcontained about 0.5 ml of air along with the powder mixture was closedwith a plug-type cap.

Just prior to use the two syringes were coupled using a double-endedconnector. The contents of the syringes were mixed during about 30reciprocations. Installation of the mixture into a wound may beaccomplished via a needle or oannula attached to the syringe containingthe mixture. The setting time for this formulation is 8-10 minutes.

Substitution of a mixture of ciprofloxacin and clindamycin (75 mg each)for ciprofloxacin yields a broad spectrum formulation covering anaerobesas well as aerobes.

Example 3 Preparation of Bupivacaine FE/CMC Matrix

Bupivacaine (100 mg) was placed into a mortar and ground with 100 mgcarboxymethylcellulose sodium (CMC) (M_(w) 70,000). Oxidized dextran(M_(w), 500,000; 66 mg/ml; 1 ml) was blended with the bupivacaine/CMCmixture at room temperature. After a homogenous suspension was obtained,adipic acid dihydrazide (7.8 mg) was added. A sample (200 mg) of theresulting gel was transferred to a 2 ml centrifuge tube for the releaseexperiment in PBS buffer. The release profile of the formulation appearsin Table 1 below:

TABLE 1 Day % Release/Day 1 10 2 10 3 10 4 9 5 5 6 5 7 5 8 3

Example 4 Preparation of Bupivacaine FB/Dextran Sulfate Sodium (DSS)Matrix

Bupivacaine FB (100 mg) was finely ground with 50 mg DSS (Mw, 500,000).Oxidized dextran solution (Mw, 500,000; 66 mg/ml; 1 ml) was added to thebupivacaine-DSS powder and mixed at room temperature. To the resultinghomogenous suspension adipic acid dihydrazide (7.8 mg) and succinic acid(10 mg) were added. A sample (200 mg) of the reaction mixture wastransferred into a 2 ml centrifuge tube for the release experiment inPBS buffer. The release profile of the matrix appears in Table 2 below:

TABLE 2 Day % Release/Day 1 11 2 10 3 9 4 7 5 7 6 7 7 6 8 4

Example 5 Preparation of Piperacillin Matrix

Piperacillin (sodium salt, 50 mg) was placed in 5 ml beaker. Oxidizeddextran solution (Mw, 500,000; 85 mg/ml; 1 ml) was added to thepiperacillin and mixed thoroughly at room temperature. After ahomogenous suspension was created, adipic acid dihydrazide (10 mg) wasadded and mixed thoroughly. A sample (200 mg) of the resultingformulation was transferred to a 2 ml centrifuge lube for the releaseexperiment in PBS buffer. The release profile of the matrix appears inTable 3 below:

TABLE 3 Day % Release/Day 1 42 2 28 3 21 4 8

Example 6 Preparation of Ceftiofur Matrix

Ceftiofur-HCl (50 mg) and adipic acid dihydrazide (10 mg) were placed ina 1-ml syringe. This syringe was connected to a second syringecontaining 1 ml of oxidized dextran (Mw, 500,000; 85 mg/ml; 1 ml). Thecontents of the two syringes were mixed by making 20 reciprocations. Asample (200 μl) of the reaction mixture was transferred to a 2 mlcentrifuge tube for the release experiment in PBS buffer. The releaseprofile of the matrix appears in Table 4 below:

TABLE 4 Day % Release/Day 1 2.5 2 2.5 3 2.9 4 3.0 5 3.2 6 3.5 7 4.0

Example 7 Preparation Azoalbumin Matrix

Sodium phosphate (40 mg) was dissolved in 1 ml of oxidized dextransolution (Mw, 500,000; 66 mg/ml). Azoalbumin (30 mg) was added and mixedthoroughly at room temperature. After homogenous suspension was createdadipic acid dihydrazide (8 mg) was added. After about one hour, 200 mgof the resulting complex was transferred into a centrifuge lube for therelease experiment in PBS buffer. The release profile of the matrixappears in Table 5 below:

TABLE 5 Day % Release/Day 1 25 2 14 3 13 4 10 5 8 6 7 7 6 8 5 9 4 10 311 3 12 3

Example 8 Formulation of Amikacin/Demineralized Bone Matrix (DBM)

Amikacin Sulfate (100 mg), DBM (200 mg), and 8 mg of adipic aciddihydrazide were placed into a beaker and mixed thoroughly. Oxidizeddextran solution (Mw, 500,000; 66 mg/ml; 1 ml) was added to the mixture.Tobramycin-sulfate or tobramycin-sulfate/clindamycin-HCl (1/1) can beused in place of amikacin-sulfate.

Example 9 Preparation of Paclitaxel Matrix

Paclitaxel (30 mg) was finely ground with 40 mg of monosodium phosphateand 30 mg adipic dihydrazide. The resulting powder was transferred to a3 ml syringe fitted with a Luer plug. The syringe was connected to asecond syringe containing 3 ml of oxidized dextran (Mw, 500,000; 65mg/ml). After about thirty reciprocations of the syringe plungers, thegel was ready to inject. The liquid mixture may therefore be installedin the cavity left by lumpectomy where it gels in situ. The paclitaxelmay kill cancer cells on the margins of the cavity and also serves as aradiation sensitizer during adjuvant radiation.

Example 10 Preparation of Vancomycin Matrix

Vancomycin (100 mg) was finely ground with 30 mg DSS (Mw, 500,000) and 8mg adipic dihydrazide. The mixture was loaded into a 3-ml syringe fittedwith a Luer plug. Oxidized dextran solution (1 ml, Mw of 500,000; 66mg/ml) was loaded into a second syringe. An injectable formulation wasformed after connecting the syringes and performing 30 reciprocations ofthe syringe plungers.

Example 11 Preparation of Doxycycline Matrix

Doxycycline-HCl (100 mg) was finely ground with 90 mg DSS (Mw, 500,000)and 8 mg adipic dihydrazide. The mixture was loaded into a 3-ml syringefitted with a Luer plug. Oxidized dextran solution (1 ml, Mw of 500,000;66 mg/ml) was loaded into a second syringe. An injectable formulationwas formed after connecting the syringes and performing 30reciprocations of the syringe plungers.

Example 12 Preparation of Antibiotic foam

One syringe contains a polymer solution-oxidized dextran (70,000 Mw, 10%oxidation, 160 mg/ml) and carboxymethylcellulose (medium viscosity, 16mg/ml). The second syringe contains 15 mg of adipic dihydrazide, 50 mgciprofloxacin, and 30 mg of an effervescent mixture, which consisted of1.2 g sodium bicarbonate and 1 g of citric acid. The syringes wereconnected and the contents mixed by 20 reciprocations. This antibioticfoam is advantageously used in treatment of wounds which are infected orare likely to become infected.

Example 13 Preparation of a Moldable Gels and Films

One syringe contains oxidized dextran (70,000 Mw, 160 mg/ml, and 10%oxidation) and carboxymethylcellulose (medium viscosity, sodium salt, 16mg/ml). The second syringe contains adipic dihydrazide (15 mg) and 50 mgciprofloxacin. The syringes were connected and the contents mixed andexpressed after 20 reciprocations. Starch glycolate (sodium salt, 30 mg)can be substituted for carboxymethylcellulose. These formulations can bequickly spread to cast a film. The alternative is to shape them after anacceptable plasticity is attained prior to installation into a defect orapplication to a wound.

Example 14 Preparation of Carboxylic Acid Salts of AminoglycosideAntibiotics

Amikacin (586 mg) was suspended in 10 ml of 80% methanol. Octanoic acid(700 μl) was added with stirring. The mixture was stirred for 3 hours atroom temperature. The solvent was evaporated and the residue wastriturated with hexane. A solid resulted with a melting point of105-110° C. Using a stoichiometry of 4 acid to 1 amikacin other saltscan be made using this procedure. Examples include hexanoate andlaurate. Similarly, hexanote, octanoate, and laurate salts oftobramycin, and gentamycin have been prepared in accordance with theprocedures of this Example 13.

Example 16 Preparation of Carboxylic Acid Salts of Clindamycin

Clindamycin (460 mg) was dissolved in 5 ml of 80% methanol. Lauric acid(200 mg) was added with stirring. The mixture was stirred for 2 hours atroom temperature and then concentrated with a rotary evaporator.Trituration with 10 ml of hexane yielded a solid. After drying in vacuoovernight the melting point was found to be 103-105° C. Other usefulsalts include hexanoate and octanoate. Lincomycin salts can be preparedin a similar fashion.

Example 16 Preparation of Carboxylic Acid Salts of Bupivacaine

Bupivacaine (576 mg) was dissolved in 7 ml of 80% methanol. To thisstirred solution was added 316 μl of caprylic acid. After two hours atroom temperature, the mixture was concentrated using a rotaryevaporator. The resulting solid had a melting range of 90-96° C.Hexanoate and laurate salts can be prepared in a similar manner.

Example 17 Preparation of Doxycycline Matrix

Doxycycline (100 mg) was finely ground and mixed with 6 mg adipicdihydrazide. This mixture was loaded into a 3-ml syringe fitted with aLuer plug. Oxidized dextran solution (1 ml, Mw of 500,000; 110 mg/ml)was loaded into a second syringe. An injectable formulation was formedafter connecting the syringes and performing 30 reciprocations of thesyringe plungers.

Example 18 Preparation of Antibiotic Coated Implants

Tobramycin caprylate (50 mg) and clindamycin caprylate (50 mg) wereground together. This solid mixture was loaded into Syringe A (3 ml)with 10 mg of adipic dihydrazide. A solution of oxidized dextran (1 ml;Mw 70,000; 10% oxidation; 100 mg/ml) was loaded into Syringe B (3 ml). Afemoral stem was pre-heated to 60° C. Syringe A and Syringe B wereconnected and the contents mixed with 20 reciprocations of the syringeplungers. Using a brush and one the syringes fitted with a bluntcannula, the implant was coated with homogenous layer of material. Afterdrying overnight, the coated implant was immersed in a tube containingphosphate buffered saline (PBS) at 37° C. The eluate showedantibacterial activity for more than one week.

While the invention has been described in connection with what ispresently considered to be the most practical and preferred embodiment,it is to be understood that the invention is not to be limited to thedisclosed embodiment, but on the contrary, is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

What is claimed is:
 1. A method of delivering a bioactive agent to abody site in need of the same, comprising the steps of: (i) providing afirst aliquot portion of a reaction mixture comprising an aldehydicpolymer solution, and a second aliquot portion of a reaction mixturecomprising a cross-linking hydrazide and a bioactive agent; (ii) mixingthe first and second aliquot portions to form the reaction mixturethereof; and (iii) installing the reaction mixture at the body site andallowing the reaction mixture to solidify in situ within about 1 to 10minutes into a reaction product comprised of a hydrazide cross-linkedoxidized aldehydic polymer matrix with the bioactive agent entrappedtherein.
 2. The method of claim 1, wherein step (i) includes providing adouble syringe having first and second syringe barrels whichrespectively contain the first and second aliquot portions of thereaction mixture.
 3. The method of claim 2, wherein step (ii) includesexpelling the first and second aliquot portions of the reaction mixturesfrom the first and second syringe barrels, respectively.
 4. The methodof claim 3, wherein step (ii) includes repeatedly transferring the firstand second aliquot portions of the reaction mixtures between the firstand second syringe barrels so as to mix the same.
 5. The methodaccording to claim 1, wherein the bioactive agent comprises at least oneselected from osteoinductive agents, antibiotics, anesthetics, growthfactors, cells, anti-tumor agents, anti-inflammatory agents,antiparasitics, antigens, adjuvants and cytokines.
 6. The methodaccording to claim 1, wherein the bioactive agent is an osteoinductiveagent which comprises demineralized bone matrix (OBM).
 7. The methodaccording to claim 1, wherein the bioactive agent is an antibiotic. 8.The method according to claim 1, wherein the antibiotic is gentamycin.9. The method of claim 1, wherein the aldehydic polymer has a molecularweight of 40,000 or greater.
 10. The method of claim 1, wherein thealdehydic polymer is oxidized dextran.
 11. The method as in claim 1,wherein the reaction mixture has a pH of 4 to
 6. 12. The method as inclaim 11, which comprises mixing a PH adjusting agent in an amountsufficient to achieve a pH of the reaction mixture of 4 to 6
 13. Themethod according to claim 1, wherein the reaction product is in the formof a gel, microsphere, film, foam, or fiber
 14. A method of deliveringan antibiotic to a body site in need of the same, comprising the stepsof: (i) providing a first aliquot portion of a reaction mixturecomprising an aldehydic polymer solution in a first syringe barrel, anda second aliquot portion of a reaction mixture comprising across-linking hydrazide and an antibiotic in a second syringe barrel;(ii) mixing the first and second aliquot portions by expelling the firstand second aliquot portions of the reaction mixtures from the first andsecond syringe barrels, respectively, to form the reaction mixturethereof; and (iii) installing the reaction mixture at the body site andallowing the reaction mixture to solidify in situ within about 1 to 10minutes into a reaction product comprised of a hydrazide cross-linkedoxidized aldehydic polymer matrix with the antibiotic entrapped therein.15. The method according to claim 14, wherein the antibiotic isgentamycin.
 16. The method according to claim 14, wherein step (ii)comprises repeatedly transferring the first and second aliquot portionsof the reaction mixtures between the first and second syringe barrels soas to mix the same.
 17. The method according to claim 16, wherein thefirst and second syringe barrels are provided by a double syringe. 18.The method of claim 14, wherein the aldehydic polymer has a molecularweight of 40,000 or greater.
 19. The method of claim 18, wherein thealdehydic polymer is oxidized dextran.
 20. The method as in claim 14,wherein the reaction mixture has a pH of 4 to
 6. 21. The method as inclaim 20, which comprises mixing a PH adjusting agent in an amountsufficient to achieve a pH of the reaction mixture of 4 to 6